Modular fluid dispensing devices

ABSTRACT

The present disclosure provides devices which deliver fluids from several reservoirs to a reaction vessel and eventually to a waste chamber in a predetermined schedule. The device provides improved simplicity while improving operational robustness and flexibility.

FIELD

The present disclosure is directed, in part, to devices that deliverfluids from several reservoirs to a reaction vessel and eventually to awaste chamber in a predetermined schedule, and to methods of carryingout the same.

BACKGROUND

In the field of analytical chemistry, there is a need for improvedliquid dispensing devices to probe samples of interest. In particular,in biochemistry a technique called Western Blot analysis requires thetimed application of at least three aqueous solutions to probe amembrane which contains samples of interest. The samples are usuallyproteins, but can also be DNA or carbohydrates. The samples are usuallyseparated into individual molecular species by SDS-PAGE electrophoresisfollowed by electroblotting on to the membrane. Direct application ofpure or complex samples has also been used by pipetting onto themembrane. The membrane binds the sample molecules due to its hydrophobicnature (PVDF membranes) or covalent crosslinking (nitrocellulosemembranes). The membrane can then be probed with various detectionprocedures which identify the particular chemical of interest. It isthis last step that requires sequential application of two to fivesolutions in a timed schedule.

There are presently four methods of probing the membranes describedabove: 1) manual application of solutions with several timed incubationsover a period of about four hours; 2) automated machines that apply asimilar routine as the manual operator; 3) a filter paper capillarydevice that applies one routine (e.g., iBind device by LifeTechnologies); and 4) a vacuum based method that moves solutions acrossa membrane by suction (e.g., SNAP id device by EMD Millipore Inc.). Allfour approaches aim to achieve a similar routine: first, block themembrane with a non-specific solution of protein, DNA, or carbohydrate,which will reduce background noise in the final result; second, in somescenarios a wash step is included; third, the specific analyticalreagent (SAR) or primary reagent is added, which binds to the desiredtarget molecule to be measured; fourth, one to six wash steps to removeexcess SAR; fifth, application of a second generic reagent thatamplifies the signal of the first; and finally, application of 1 to 6washes with buffer to remove excess reagent. The membrane is then readyfor signal development in colorimetric, fluorescence, radiometric, orluminescence modes.

This six step procedure is very time consuming and labor intensive. Theoperator has to return to the membrane once per hour in the reagentincubation steps and every 3 to 5 minutes in the wash steps, for a totalof between 10 and 17 times. In addition, different operators prefer torun different procedures. For example, a first operator may desire touse 3×5 washes and a 2 hour SAR step and a 1 hour secondary reagentstep, whereas a second operator might use a 16 hour SAR and only onewash step.

The machines that have been developed to date allow a user to controlwash times and reagent incubation times and address the need forautomation. However, the machines are highly complex with variouscombinations of pumps, pressurized air supply and/or actuators, andrequire a trained individual to operate them. For example U.S. Pat. Nos.4,859,419, 8,404,198, 5,567,595, 5,559,032, 6,194,160, 4,585,623, and8,758,687, as well as iBind machine (internet at:tools.lifetechnologies.com/content/sfs/manuals/ibind_man.pdf) and theSnap id machine (internet at:emdmillipore.com/US/en/life-science-research/protein-detection-quantification/SNAP-i.d.-2.0-Protein-Detection-System-/snap-id-system-western-blotting/m9Wb.qB.wzoAAAFBrt8RRkwt.nav)report various machines, some of which are complicated and/or inflexibleand require trained personnel to operate and maintain, which makesoperation and repair expensive. Thus, there is a need to reducecomplexity and costs of manufacture.

The present disclosure addresses many of the drawbacks of the presentlyavailable devices.

SUMMARY

The present disclosure provides liquid dispensing devices comprising: awash buffer reservoir and at least one reagent reservoir, each reservoircomprising a reservoir control valve, wherein each reservoir controlvalve is in electrical communication with an electronic control board; areaction vessel in fluid communication with the wash buffer reservoirand the at least one reagent reservoir, wherein the reaction vesselcomprises one or more baffles; a waste tray in fluid communication withthe reaction vessel; a motorized cam in physical connection with thereaction vessel; and an optional central pump as described herein.

The present disclosure also provides systems comprising a plurality ofdevices as described herein.

The present disclosure also provides methods of analyzing a membraneusing any of the devices described herein comprising: loading a washbuffer into the wash buffer reservoir; loading a first reagent into oneof the at least one reagent reservoirs; optionally loading a secondreagent into a different one of the at least one reagent reservoirs;loading the membrane and blocking reagent into the reaction vessel;selecting a routine from the electronic control board; and removing themembrane from the reaction vessel for subsequent signal development andresult acquisition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a representative CAD 3D schematic of a device.

FIG. 2 shows a representative device without a chassis cover revealingseveral internal components.

FIG. 3 shows a representative platform of a device that supports areaction vessel (which has been removed from the view).

FIG. 4 shows an underneath view of a representative device.

FIGS. 5A and 5B show representative cam designs and the reaction vesselwave indicating asymmetric and symmetric two point cam design.

FIGS. 6A, 6B, and 6C show a first representation of a reaction vesseldesign.

FIGS. 7A, 7B, and 7C show a second representation of a reaction vesseldesign.

FIGS. 8A, 8B, and 8C show a third representation of a reaction vesseldesign.

FIG. 9 shows a representative electrical layout of a device, indicatinga simplistic design of a control board, valves, and motor control.

FIG. 10 panel A and FIG. 10 panel B show a representative flow chart ofstandard operating procedure for using a device, and summary of stepsand times for programming, respectively.

FIG. 11 shows a representative image of a PVDF-membrane, processed usinga device described herein, which was placed into a dark camera chamber.

DESCRIPTION OF EMBODIMENTS

A goal of the present disclosure was to create a low cost alternative toavailable devices which are flexible in procedures but complicated toprogram and expensive to purchase, operate and maintain. To realize thisgoal, a minimalist approach was taken to the design of a plumbing schemeof valves and pumps, utilizing gravitational potential or a central pumpto propel liquids through valves and tubes. Due to its simplicity,feedback loops from sensors are not required to maintain robustoperation and minimizing components allows the foot print to bedramatically reduced compared to other devices (e.g., U.S. Pat. Nos.4,859,419, 8,404,198, 5,567,595, 5,559,032, 6,194,160, 4,585,623, and8,758,687, as well as iBind machine and the Snap id machine. Inaddition, by utilizing advanced low friction plastics, the designsimplifies the method of agitating a membrane.

The device, when configured without a central pump, can be divided intofour gravitational levels. The highest level contains the wash reservoirand the at least one reagent reservoir. The second level is at theheight of the reservoir control valves. The third level is at thereaction vessel height. The fourth level is where the waste isdeposited. By purposely designing these levels with similar heights itis possible to create a reliable flow of liquid between any two stageswhile stopping and starting the flow with a valve control.

The present disclosure provides liquid dispensing devices. The liquiddispensing devices comprise a wash buffer reservoir, at least onereagent reservoir, multiple reservoir control valves, an optionalcentral pump, an electronic control board, a reaction vessel thatcomprises one or more baffles, a waste tray, and a motorized cam. FIG. 1shows a representative liquid dispensing device having a wash bufferreservoir (1), reagent reservoirs (2 and 3), waste tray (24), chassis(25), and a reaction vessel (5). FIG. 2 shows a representative liquiddispensing device showing a wash buffer reservoir (1), reagentreservoirs (2, 3), a reaction vessel (5), a first port of the reactionvessel (6), wash buffer reservoir port (26), reagent reservoir ports(27), and a reaction vessel control valve (23). FIG. 3 shows arepresentative liquid dispensing device in which the reaction vessel hasbeen removed showing a geared motor (7), cam (8), and reaction vesselclips (9). FIG. 4 shows the underside of a representative liquiddispensing device showing four reservoir control valves (4), a gearedmotor (7), a cam (8), a multi-way connector (12), tubing connectingreaction vessel and waste tray (28), and various connection tubing (13)and wiring.

In some embodiments, the wash buffer reservoir (1) serves as a chamberinto which a wash buffer is poured. Any wash buffer can be used. Thewash buffer reservoir can be of any shape and size, but is generallydesigned to hold from about 50 ml to about 500 ml, from about 100 ml toabout 400 ml, or from about 200 ml to about 300 ml of liquid. The washbuffer reservoir also comprises a port (not shown), which serves as anopening into which the wash buffer can leave the wash buffer reservoir.The port can be located anywhere in the bottom of the wash bufferreservoir. In some embodiments, the port is located on the bottom of thewash buffer reservoir at one end of the reservoir. In some embodiments,the wash buffer reservoir is designed to be removable and allow completedrainage by gradients in the floor of the tray.

In some embodiments, the device comprises at least one reagent reservoir(2, 3). In some embodiments, the device comprises two or three reagentreservoirs (i.e., a primary reagent reservoir and a secondary reagentreservoir). Each reagent reservoir serves as a chamber into whichvarious reagents are poured. Any reagent can be used. Suitable reagentsinclude, for example, primary and secondary antibodies used fordetection. The reagent reservoirs can be of any shape and size, but aregenerally designed to be at least 1 cm, at least 2 cm, or at least 4 cmin height, and/or designed to contain up to 0.5 ml, up to 2 ml, up to 5ml, up to 10 ml, up to 20 ml, or up to 50 ml of reagent. Each reagentreservoir also comprises a port (not shown), which serves as an openinginto which the reagent can leave the reagent reservoir. The port can belocated anywhere in the bottom of the reagent reservoir. In part becausethe primary and secondary reagents are generally expensive, and thussmall volumes (1 to 10 ml) are usually employed.

In some embodiments, the device comprises a reaction vessel (5) whichserves as a chamber into which a membrane is placed. Any membrane,including PVDF and nitrocellulose membranes, can be used. The reactionvessel may also be used to contain various buffers, such as a blockingsolution, that are added for a particular protocol. The reaction vesselcan be of any shape and size, but is generally designed to hold a squareor rectangular piece of membrane that is from about 5 cm to about 30 cm,from about 8 cm to about 20 cm, or from about 10 cm to about 15 cm insize. The reaction vessel is also designed to hold from about 0.5 ml toabout 100 ml, from about 2 ml to about 75 ml, from about 5 ml to about20 ml, or from about 8 ml to about 20 ml of liquid. In some embodiments,the reaction vessel comprises one or a plurality of baffles. The bafflesmay be of any shape and may be of any length to fit inside the reactionvessel, each baffle being substantially parallel to one or more otherbaffles. The reaction vessel further comprises a first port (6) (see,FIG. 2), which serves as an opening into which any fluids can enter thereaction vessel from the either or both the wash buffer reservoir and/orthe reagent reservoir(s). The first port can be located anywhere in theside of the reaction vessel or may simply be the opening of the reactionvessel at the top. In some embodiments, the reaction vessel comprises asecond port (22) (see, FIGS. 6A, 6B, 6C, 7A, 7B, 7C, 8A, 8B, and 8C),which serves as an opening in the reaction vessel into which any fluidsthat are within the reaction vessel can exit the reaction vessel. Insome embodiments, the second port of the reaction vessel is located onthe bottom of the reaction vessel at one end of the reaction vessel. Thebaffles also retain the membrane in the fluid area while stopping itfrom obstructing the second port in the reaction vessel. In someembodiments, the reaction vessel is asymmetric in regard to the secondport and axis of rotation of the reaction vessel. In some embodiments,the reaction vessel comprises a plurality of analysis sections, whichcan each comprise a separate membrane. The reaction vessel can be madeout of low friction plastic or a low friction plastic strip is adheredto its bottom base to interact with the cam. The reaction vessel designis not limited to that in any of the figure disclosed herein; otherdesigns are possible which may have smaller or larger surface area andvolume, or contain a cylinder of material that acts as a dispersalmechanism for low (<5 ml) of liquid, or contain multiple chambers forincubating membranes of different size or number. In some embodiments,the reaction vessel can be held in place by one or more reaction vesselclips (9) (see, FIG. 3).

The wash buffer reservoir and the at least one reagent reservoir areeach in fluid communication with the reaction vessel. Thus, fluids mayflow from the wash buffer reservoir to the reaction vessel, and can doso merely by gravitational forces in devices without a central pump.Likewise, fluids may flow from the at least one reagent reservoir to thereaction vessel, and can do so merely by gravitational forces in deviceswithout a central pump. Fluids leave the wash buffer reservoir and theat least one reagent reservoir through their respective ports, asdescribed above. The fluid(s) leaving the wash buffer reservoir and/orthe at least one reagent reservoir enter the reaction vessel through thefirst port of the reaction vessel. Each port of the wash bufferreservoir and the at least one reagent reservoir are in fluidcommunication via tubing with the first port of the reaction vessel. Insome embodiments, the fluid communication is carried out by tubing. Insome embodiments, each of the tubing is about ⅛ inch internal diameterand about 3/16 inch outside diameter. Any tubing can be used. In someembodiments, the tubing is smoothly contoured so that resistance is notincreased by kinks. FIG. 4 shows tubing (13) connecting one portion ofthe device to another portion of the device.

The flow of fluid from either or both of the wash buffer reservoir andthe at least one reagent reservoir to the reaction vessel is controlledwith one or more reservoir control valves (4), and the optional centralpump (27). The reservoir control valve for the wash buffer reservoir maycontrol the opening and closing of the port of the wash bufferreservoir. Alternately, while the port of the wash buffer reservoir mayalways be open, the reservoir control valve for the wash bufferreservoir may be an in-line component of the tubing from the wash bufferreservoir to the reaction vessel, and thus control the flow of fluidonce in the tubing. Likewise, the reservoir control valve(s) for the atleast one reagent reservoir(s) and the optional central pump may controlthe opening and closing of the port(s) of the reagent reservoir(s).Alternately, while the port of the reagent reservoir(s) may always beopen and the optional central pump operating, the reservoir controlvalve for the at least one reagent reservoir(s) may be in-linecomponent(s) of the tubing from the at least one reagent reservoir tothe reaction vessel, and thus control the flow of fluid once in thetubing. The optional central pump is in electronic communication withthe control board and can act to pull or push fluid through the variousports and tubing in the device. The central pump may also be incommunication with a power source.

In some embodiments (such as, but not limited to, devices without acentral pump), the port of the at least one reagent vessel and the portof the wash buffer reservoir are both at least 0.5 cm higher, at least 1cm higher, at least 2 cm higher, at least 3 cm higher, at least 4 cmhigher, or at least 5 cm higher than each of the respective reservoircontrol valves. In some embodiments, each of the reservoir controlvalves are at least 0.5 cm higher, at least 1 cm higher, at least 2 cmhigher, at least 3 cm higher, at least 4 cm higher, or at least 5 cmhigher than the first port of the reaction vessel.

In some embodiments, the tubing leaving the respective reservoir controlvalves (i.e., tubing from the wash buffer reservoir leading to itsreservoir control valve, and the tubing from each of the reagentreservoir(s) leading to its/their reservoir control valve(s); andleaving the reservoir control valves) converge to a multi-way connector(12) (see, FIG. 4). The multi-way connector is in fluid communicationwith the optional central pump and reaction vessel by additional tubingwhich enters the reaction vessel by the first port of the reactionvessel or simply disposes its contents into the opening for the reactionvessel. In some embodiments, the multi-way connector is a 4-wayconnector. In some embodiments, the multi-way connector that receivesthe reservoir solutions is mounted at an angle and close to the reactionvessel. In this manner, there is a lower probability of crosscontamination between different solutions for different steps in theprocedures because the tubing connecting the multi-way connector and thereaction vessel is short and it is tilted downward. Whichever fluid isflowing through the connector will continue to flow until all fluid hasmoved into the reaction vessel because the connector, in particular theportion of the connector leading to the reaction vessel, is angleddownwards into the reaction vessel.

In some embodiments, the waste tray serves as a chamber into which thefluid from the reaction vessel drains. The waste tray can be of anyshape and size, but is generally designed to hold from about 100 ml toabout 2000 ml, from about 150 ml to about 1500 ml, from about 200 toabout 800 ml, or from about 250 ml to about 400 ml of fluid. In someembodiments, the waste tray comprises a port, which serves as an openinginto which the fluid from the reaction vessel (through the second portthereof) can enter the waste tray. The port can be located anywhere inthe top portion of the waste tray. The waste tray is in fluidcommunication with the reaction vessel by tubing. In some embodiments(such as, but not limited to, devices without a central pump), thesecond port of the reaction vessel is at least 0.5 cm higher, at least 1cm higher, at least 2 cm higher, at least 3 cm higher, at least 4 cmhigher, or at least 5 cm higher than the port in the waste tray.

In some embodiments, the flow of fluid from the reaction vessel to thewaste tray reaction is controlled with a reaction vessel control valve(23) (see, FIG. 2). The reaction vessel control valve may control theopening and closing of the second port of the reaction vessel.Alternately, while the second port of the reaction vessel may always beopen, the reaction vessel control valve may be an in-line component ofthe tubing from the reaction vessel to the waste tray, and thus controlthe flow of fluid once in the tubing.

The devices further comprise a cam (8), powered by a geared motor (7),in physical connection with the reaction vessel. The cam is designed totilt the reaction vessel from one to four times, or twice, for everyrotation of the motor output. The cam can also use 1, 2, 3, 4, 5 or morehigh points for tilting. The motor output rotates at about 40 rpm with12 V power and, therefore, the tilting up and down cycle is performedtwice per revolution and 80 times per minute, but the frequency is notlimited to this rate and can also be higher or lower. The cam can bemade out of low friction plastic which interacts with the underside ofthe reaction vessel which can also be interfaced with low frictionplastic and, thus, create a low friction tilting mechanism. In someembodiments, the cam comprises an off-set shape. In some embodiments,the reaction vessel is tethered at one end and is positioned to rock upand down at the distal end (i.e., non-tethered end) by interaction withthe cam. Thus, in some embodiments, the reaction vessel is designed totilt back and forth through a small angle with an axis on one end of thevessel. The tilting amplitude is helpful for a good waveform to maintainappropriate liquid movement across the membrane which improves detectionof analytes and reduces background signals. In some embodiments, thereaction vessel further comprises a cylinder that moves up and down withthe action of the cam. A simple geared motor with low friction plasticcam creates a low friction tilting mechanism which is programmed to beon at the beginning of the routine and off at the end. Thus, an on/offcontrol switch for tilting need not be required. The motor is inelectronic communication with the control board and with a power source(not shown).

FIGS. 5A and 5B show representative cam designs and the reaction vesselwave indicating asymmetric and symmetric two point cam design. Each ofthe cams can be from about 2 to about 20 mm in width and can have avariety of radius designs from which to select, depending upon thedegree of tilt desired for the reaction vessel. The asymmetric designcreates good drainage and a good waveform by spending more time in thetilted up position and a rapid drop to the lowest level which creates awave in the liquid which can be desirable compared to constant tiltingwithout a wave. The asymmetric cam can be made in various shapes suchas: the two high points having a radius which is 3 mm wider than thesecond highest point which is at right angles to them, the secondhighest point having a radius which is 2 mm wider than the lowest pointwhich is opposite the second highest point. If the reaction tray isconsidered in a reference location when it is horizontal (no tilt), thenthe high point of the tilt is 9 mm above horizontal, the second highpoint is 2 mm above horizontal and the lowest point is 2 mm belowhorizontal. The design of the cam allow these different tilt amplitudesto occur. Other radii can be used to create different tilting amplitudesand frequencies.

FIGS. 6A, 6B, 6C, 7A, 7B, 7C, 8A, 8B, and 8C show representative designsfor the reaction vessel and how it interacts with other components inthe liquid dispending device. In some embodiments, the introduction offluid is from the multi-way connector which directs fluid into thereaction vessel from above (where the first port is actually the openingto the reaction vessel). The membrane and fluid are agitated in thereaction vessel by tilting, and the baffles keep the membrane out of theway of the second port drain which is located at the axis of rotation.Because of the cam shape, the reaction vessel can be tilted up at itsdistal end for the majority of the tilting cycle, which aids thedrainage of fluid to the waste tray.

Referring to FIGS. 6A, 6B, and 6C, a first reaction vessel design isshown. The basic design has a flat lower surface with baffles (14)separating the membrane probing area (15) from the second port (22) ofthe reaction vessel, and the axis of rotation is in the location of thetray retaining clips next to the chassis bulk head end. The tray tiltsdown only 1 or 2 mm at its distal end and upward by 8 to 10 mm. Othertilting angles and amplitudes are possible and work well. In someembodiments, the reaction vessel can comprise one or more clips in legs(17), as shown on the bulk head end. Other reaction vessel designs arepossible which allow for smaller volumes of SAR, different sizes andnumbers of membranes (e.g., the use of a plastic cylinder as a dispersalmechanism for small volumes (see, FIGS. 7A, 7B, and 7C) or using, forexample, fifteen mini-chambers for incubating strips of 0.3 cm×8 cmwithin one reaction vessel (see, FIGS. 8A, 8B, and 8C).

Referring to FIGS. 7A, 7B, and 7C, a second reaction vessel design isshown. The small volume of SAR is necessary in some cases where SAR islimiting (e.g., with expensive antibody solutions or when using mouseserum). This design incorporates a plastic cylinder (19) which issuitably heavy and of sufficient diameter to roll up and down thereaction vessel on top of the membrane when the vessel is inclined upand down. Liquid volumes as low as 1 ml of SAR can be evenly spreadacross a normal sized membrane thus saving valuable reagent. In someembodiments, separators (18) are located at the distal end to stopsurface tension causing the cylinder to adhere to the wall of thevessel.

Referring to FIGS. 8A, 8B, and 8C, a third reaction vessel design isshown. In some situations, the operator may need to measure the activityof antibodies in very small volumes (e.g., 200 μl or less). In thiscase, the reaction vessel has individual mini-chambers separated bywalls (20), and optionally a dam (21) to stop the SAR solution movingout of a mini-chamber and mixing with the others. During a wash step, alarge volume (e.g., 20 ml or more) of wash buffer flows over the dam andinto the whole reaction vessel and immediately drains out of the secondport, thus not allowing time for SARs to contaminate each other'schamber.

In each of the embodiments disclosed herein, each control valve is inelectrical communication with an electronic control board (10) (see,FIG. 9), which comprises a start button (26), which can be utilized tostart a particular routine, and a rotary switch (25), which can be usedto select a particular routine. Thus, the electronic control boardcontrols the opening and closing of each control valve (4) (see V1, V2,V3, V4, P1 in FIG. 9) and fluid pumping and controls the length of timethat each control valve is opened, and thus the volume of fluid beingtransported. In some embodiments, the electronic control board iscommercially available, such as, for example, Arduino Uno or Mega, orthe like. In some embodiments, one or more of the control valves may beoperated manually. In some embodiments, the electronic control boardcontains software that is programmable to carry out particular routinesto control the sequence of control valve opening and closing and thelength of time each control valve is opened. In some embodiments, thecontrol board will have a power switch to control the operation of themotor for the cam. In some embodiments, the electronic control board isprogrammable to control the speed of the motorized cam. In someembodiments, the electronic control board further comprises acommunication port, such as a USB port for ISO9000 type compliance, foranother device. In some embodiments, the USB port is included forISO9000-type reporting of the device's events during a particularprocedure. In some embodiments, the electronic control board comprisesBluetooth remote control capability, which can allow for remoteprogramming and control of the device.

The control software program for the control board is very flexible withall steps being able to be modified and reprogrammed. However, a shortlist of routines can cover a majority of practical procedures. Thus, abasic program design which incorporates this short list can simplify theinterface with the end user by providing several push buttons or arotary switch with several positions. For example, one routine providesthe opportunity to treat the membrane with just two reagents (e.g., washand primary reagent) which gives flexibility for other applications suchas probing with a pre-labeled primary reagent. The pre-programmedroutines can be varied in time and number of steps.

The devices disclosed herein can be compact and portable. In someembodiments, the entire device is less than 30 cm×50 cm, less than 20cm×30 cm, or less than 16 cm×24 cm, and weighs less than 10 kg, or lessthan 5 kg, or less than 2 kg. In some embodiments, the device is about15 cm×about 22 cm. In some embodiments, a chassis connects each of thewash buffer reservoir, reagent reservoir(s), reaction vessel, wash tray,and cam motor and is also the exterior shell of the device. In someembodiments, the device comprises rounded corners, high sloping walls,and/or lower walls that are mainly vertical to allow close packing.

The reaction vessel is tilted with an asymmetric amplitude to create awaveform in the fluid which can be adjusted, by placement of the camfrom a centered position to any one of a number of off-centerplacements. This allows for agitation and improved analyte detection,and also aids the draining of fluids at the end of each step.

The present disclosure also provides a system that comprises a pluralityof any one or more of the devices disclosed herein. The system may becontrolled by a single electronic control board which controls allvalves, motors, and optional central pumps, of each device.

The present disclosure also provides methods of analyzing a membraneusing a device as described herein. In some embodiments, such as aWestern Blot embodiment, a wash buffer is loaded into the wash bufferreservoir. A first reagent is loaded into one of the at least onereagent reservoirs. A second reagent is optionally loaded into adifferent one of the at least one reagent reservoirs. The membrane andblocking reagent are placed into the reaction vessel. A routine from theelectronic control board is selected and runs for a predetermined amountof time. The membrane is removed from the reaction vessel for subsequentsignal development and result acquisition. For example, the operatorwill previously have an analyte bound-membrane from a Western Blotexperiment, and will have prepared four solutions: 1) blocking reagent(20 ml), 2) wash buffer (300 ml), 3) SAR (5 ml), and 4) secondaryamplifying reagent (15 ml). The operator will add the wash buffer intothe wash buffer reservoir, and the primary and secondary reagents intotheir appropriate reagent reservoirs. Then the membrane is placed in thereaction vessel and the blocking solution is poured over it. The desiredroutine is chosen by pressing a switch and the routine runs withoutinterruption. The operator can return after about 4 hours (or when theroutine ends) or the full length of time of another routine and removesthe membrane for subsequent signal development and result acquisition byany of the known methods. The operator can remove excess wash bufferremaining in the wash buffer reservoir and apply nanopure water to anyof the reservoirs and run a programmed wash routine which preserves thecomponents for future use.

Referring to FIG. 10A, a representative flow chart of standard operatingprocedure with variable aspects is shown. The flow chart representstypical steps in a standard operating procedure. For example, a blockedmembrane is washed (30), incubated with primary reagent (35), washed(40), incubated with secondary reagent (45), and washed again (50). FIG.10B shows a typical range of variations in Western blots and anexemplary routine programmed into the controller.

In order that the subject matter disclosed herein may be moreefficiently understood, examples are provided below. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting the claimed subject matter in anymanner.

EXAMPLES Example 1 Western Blot (Actual Example)

A typical western blot procedure was performed. First, a membrane wasprepared which contained the adhered proteins, which were separated byelectrophoresis using an SDS containing polyacrylamide gel. Severalprotein containing samples were prepared; ten lanes were loaded with: 1)molecular weight marker (MWM) (lane 1); 2) various amounts of purifiedrecombinant RhoA protein (RH01) (lanes 2 to 7); and 3) various amountsof platelet cell protein extract (lanes 8, 9, and 10). Electrophoresiswas performed at 150V for 1.5 hours. The gel was placed into blottingbuffer containing 25 mM Tris base, 180 mM glycine, and 15% methanol innanopure water to equilibrate. The gel was then sandwiched betweenfilter paper and the membrane on the positively charged side of the gel(SDS surrounded proteins migrate towards the positive terminal) andelectroblotting was performed at 350 mA for 45 minutes. The membrane wastransferred to a device as set forth herein and the following protocolwas carried out.

Four solutions were prepared: 1) Phosphate Buffered Saline plus Tween 20(PBST): 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄, 0.20 g KCl, 8 g NaCl, 995 mlnanopure water; mixed until dissolved and pH to 7.4 with 1M HCl; 2)Blocking Solution: 1 g (5% w/v) dry milk powder in 20 ml of PBST; 3)Primary Antibody in PBST: 5 μl of anti-RhoA (Cat. # ARH04 fromCytoskeleton Inc., CO, USA) in 5 ml of PBST; and 4) Secondary Antibody(horse radish peroxidase labeled, HRP) in PBST: 1 μl of anti-mouse IgGHRP (Cat. #115-035-164 from Jackson Immunoresearch Labs, PA, USA) in 20ml of PBST.

The device was turned on and the rotary knob turned to the protocolnumber 1 which is a normal routine for Western Blots. All valves were inthe closed position at the start of the routine. Three hundred (300) mlof PBST was poured into the wash buffer reservoir. Twenty (20) ml ofblocking solution was poured into the reaction vessel and the membranewas added to this solution. The primary antibody (in 5 ml solution) waspoured into the first reagent reservoir, and the secondary antibody (in20 ml solution) was poured into a second reagent reservoir. The routinewas started by pressing the start button.

About four hours later, the membrane was removed from the reactionvessel and placed on a paper towel. The membrane was dabbed dry andplaced on a piece of 4 mil polythene, to which 1 ml of HRP detectionreagent (e.g. Cat. #34076 from ThermoFisher Scientific Inc., MA, USA)was pipetted on to the membrane to develop the chemiluminescent signal.Another layer of polyurethane was sandwiched on top. Thepolyurethane-membrane sandwich was placed into a dark camera chamber andan image was taken (see, FIG. 11 for results). Detection of purifiedrecombinant RhoA at 28 kDal molecular weight with a 10 ng detectionlimit was observed at a 30 second exposure, and down to 5 ng at a 5minute exposure. The detection of native RhoA at 21 kDal molecularweight was observed where approximately 10 ng of RhoA was detected in 40μg of platelet cell extract. The dark background was indicative of verygood washing of the membrane between incubation steps.

Routine Steps:

The first versions contains what the operator sees, whereas the secondversion contains the software program valve timings.

Physical Routine

1. Tilt mechanism starts;

2. Block for one hour;

3. Wash in PBST once for 3 min;

4. Primary reagent for one hour;

5. Wash in PBST for 5 min (repeat for 3 times total);

6. Secondary reagent for one hour;

7. Wash in PBST for 5 min (repeat for 5 times total);

8. Remove membrane and develop signal with chemiluminescence substrate.

Software Routine Valve Commands

Starting Time Routine 1 (1-1-1) 00:00:00.0 Motion platform On and OpenV1(drain) 00:00:00.0 Wait for 3600 sec. 01:00:00.0 Open V1 (drain) for10.0 sec. 01:00:10.0 Close V1 01:00:11.0 Open V2 (wash) and optionalpump (P1) on for 12.0 sec. 01:00:23.0 Close V2 and P1 off 01:00:24.0Wait for 180 sec. 01:03:24.0 Open V1 (drain) for 10.0 sec. 01:03:34.0Close V1 01:03:35.0 Open V3 (primary antibody) and optional pump (P1) onfor 10.0 sec. 01:03:45.0 Close V3 and pump off 01:03:46.0 Wait for 3600sec. 02:03:46.0 Open V1 (drain) for 10.0 sec. 02:03:56.0 Close V102:03:57.0 Open V2 (wash) and optional pump (P1) on for 14.0 sec.02:04:11.0 Close V2 and pump off 02:04:12.0 Wait for 300 sec. 02:09:12.0Open V1 (drain) for 10.0 sec. 02:09:13.0 Close V1 02:09:14.0 Open V2(wash) and optional pump (P1) on for 14.0 sec. 02:09:28.0 Close V2 andpump off 02:09:29.0 Wait for 300 sec. 02:14:29.0 Open V1 (drain) for10.0 sec. 02:14:39.0 Close V1 02:14:30.0 Open V2 (wash) and optionalpump (P1) on for 14.0 sec. 02:14:44.0 Close V2 and pump off 02:14:44.0Wait for 300 sec. 02:19:44.0 Open V1 (drain) for 10.0 sec. 02:19:54.0Close V1 02:19:55.0 Open V4 (secondary antibody) and optional pump (P1)on for 10.0 sec. 02:21:05.0 Close V4 and pump off 02:21:05.0 Wait for3600 sec. 03:21:05.0 Open V1 (drain) for 10.0 sec. 03:21:15.0 Close V103:21:16.0 Open V2 (wash) and optional pump (P1) on for 17.0 sec.03:21:33.0 Close V2 and pump off 03:21:33.0 Wait for 300 sec. 03:26:33.0Open V1 (drain) for 10.0 sec. 03:26:43.0 Close V1 03:26:44.0 Open V2(wash) and optional pump (P1) on for 17.0 sec. 03:27:01.0 Close V2 andpump off 03:27:02.0 Wait for 300 sec. 03:32:02.0 Open V1 (drain) for10.0 sec. 03:32:12.0 Close V1 03:32:13.0 Open V2 (wash) and optionalpump (P1) on for 17.0 sec. 03:32:30.0 Close V2 and pump off 03:32:30.0Wait for 300 sec. 03:37:30.0 Open V1 (drain) for 10.0 sec. 03:37:40.0Close V1 03:37:41.0 Open V2 (wash) and optional pump (P1) on for 17.0sec. 03:37:58.0 Close V2 and pump off 03:37:58.0 Wait for 300 sec.03:42:58.0 Open V1 (drain) for 10.0 sec. 03:43:08.0 Close V1 03:43:09.0Open V2 (wash) and optional pump (P1) on for 17.0 sec. 03:43:26.0 CloseV2 and pump off 03:43:26.0 Wait for 300 sec. 03:48:26.0 Routine 1(1-1-1) finished.Closed all Valves and Turned Motion Platform Off

Various modifications of the described subject matter, in addition tothose described herein, will be apparent to those skilled in the artfrom the foregoing description. Such modifications are also intended tofall within the scope of the appended claims. Each reference (including,but not limited to, journal articles, U.S. and non-U.S. patents, patentapplication publications, international patent application publications,and the like) cited in the present application is incorporated herein byreference in its entirety.

What is claimed is:
 1. A liquid dispensing device comprising: a washbuffer reservoir and at least one reagent reservoir, each reservoircomprising a reservoir control valve, wherein each reservoir controlvalve is in electrical communication with an electronic control board; areaction vessel in fluid communication with the wash buffer reservoirand the at least one reagent reservoir, the reaction vessel comprisesone or more baffles; a waste tray in fluid communication with thereaction vessel; a motorized, off-set asymmetric cam in physicalconnection with the reaction vessel configured to cause a tiltingmotion; a cylinder disposed inside the reaction vessel and notphysically attached to any other components of the device in order tomove inside the reaction vessel along with the tilting motion of thecam; and a central pump in electronic communication with the electroniccontrol board and in fluid communication with the wash buffer reservoirand the at least one reagent reservoir; wherein each of the wash bufferreservoirs and at least one reagent reservoir comprise a port which isin fluid communication with a first port in the reaction vessel; whereineach of the port of the wash buffer reservoir and the port of the atleast one reagent reservoir are in fluid communication by tubing withthe first port of the reaction vessel; and wherein the tubing from thewash buffer reservoir and the tubing from the at least one reagentreservoir are in fluid communication with a multi-way connector and thecentral pump.
 2. The device of claim 1, wherein each reservoir controlvalve either controls the ports of the wash buffer reservoir and the atleast one reagent reservoir, or controls the fluid communication withinthe respective tubing.
 3. The device of claim 1, wherein the multi-wayconnector is in fluid communication with the reaction vessel by tubing.4. The device of claim 1, wherein a port in the waste tray is in fluidcommunication with a second port in the reaction vessel by tubing. 5.The device of claim 4, wherein a reaction vessel control valve eithercontrols the second port of the reaction vessel, or controls the fluidcommunication within the tubing from the reaction vessel to the wastetray.
 6. The device of claim 1, wherein the electronic control boardcontrols the opening and closing of each control valve, turns thecentral pump on and off, and controls the length of time that eachcontrol valve is opened.
 7. The device of claim 6, wherein theelectronic control board contains software that is programmable tocontrol the sequence of control valve opening and closing and the lengthof time each control valve is opened.
 8. The device of claim 1, whereinthe electronic control board is programmable to control the speed of themotorized cam.
 9. The device of claim 1, wherein the reaction vessel istethered at one end and is positioned to rock up and down at the distalend by interaction with the cam.
 10. The device of claim 1, furthercomprising a communication port for ISO9000 compliance.
 11. The deviceof claim 1, wherein the electronic control board comprises Bluetoothremote control capability.
 12. The device of claim 1, wherein thereaction vessel is asymmetric in regard to the second port and axis ofrotation of the reaction vessel, wherein the axis of rotation of thereaction vessel is at the same end of the reaction vessel as the secondport.
 13. The device of claim 1, wherein the reaction vessel comprises aplurality of analysis sections.
 14. The device of claim 1, wherein theentire device is less than 16 cm×24 cm, and weighs less than 2 kg. 15.The device of claim 1, wherein each of the tubing is about ⅛ inchinternal diameter and about 3/16 inch outside diameter.
 16. The deviceof claim 1, wherein each of the at least one reagent reservoirscomprises an interior capable of holding up to 20 ml of reagent.
 17. Thedevice of claim 1, wherein a chassis connects each of the wash bufferreservoir, reagent reservoir(s), reaction vessel, wash tray, and cammotor and is also the exterior shell of the device.